**3.2 H2S elimination from the biogas stream using a biofiltration system**

A characterization of the three potential biofilter packing materials was performed. The highest water retention capacity (WRC) was found in vermiculite (65%), while the WRC for lava rock was 15%. Although vermiculite showed a higher WRC, lava rock favored water irrigation, which ensured that the desired moisture level was maintained and avoided sulfate accumulation at the same time.

The acetic, propionic, butyric and valeric acid could also be present in the biogas stream, their assimilation was determined in batch experiments. The biodegradation of different loadings of acetic and propionic acids as individual substrates were also evaluated in the lava rock biofilter as it as previously described elsewhere (Ramirez-Saenz et al., 2009).

As the MR proportion increased in the ADS, the H2S concentration in the biogas stream also increased. The biodegradation of H2S was determined in the lava rock biofilter under two different empty-bed residence times (EBRT). Results for H2S elimination capacity as a function of H2S inlet loading in the lava rock biofilter, operated at 85 sec and 31 sec EBRT, are depicted in Figure 2. As shown in Figure 2A, at an EBRT of 85 sec, the relationship between the inlet loading and the elimination capacity was linear, and the critical H2S elimination capacity defined by Devinny et al., 1999 (i.e., deviation from the 100% removal capacity) was not yet reached at an inlet loading of 144 g/m3h. Under these operation conditions, the removal efficiency of H2S for loadings between 36 and 144 g/m3h was always above 98 %. Furthermore, the EC reached a maximum of 142 g/m3h when the H2S loading was 144 g/m3h.

For an EBRT of 31 sec (Figure 2B), the H2S elimination capacity was found to be linear with respect to H2S inlet loading up to 200 g/m3h (100% removal efficiency). A higher inlet loading of 300 g/m3h reduced the removal efficiency in the system to 85 %. An inlet loading of 400 g/m3h (corresponding to 3000 ppmv) caused the removal efficiency to drop to 75%, which suggested inhibition of biological activity and/or insufficient mass transfer. In this case, the critical H2S EC was 200 g/m3h, whereas a maximum H2S EC value of 232 g/m3h was achieved in the biofilter. At the same time, however, the removal of VFAs present in the gaseous stream (approximately 10 ppmv) reached 99%.

Biogas Production and

Cleanup by Biofiltration for a Potential Use as an Alternative Energy Source 125

a

b

c

d

Fig. 3. Biofiltration system coupled to an anaerobic digestion system. Arrows indicate the

Additionally, the changes in the bacterial community were also determined by taking samples during long-term operation of the biofiltration system (Samples from 1a to 9a). The analysis was performed by a DDGE system using 16S rRNA as a bacteria-specific target for PCR amplification. Figure 4 shows an example of denaturing gradient gel DDGE (15% to 60%) from samples of different times of cultivation compared with the initial bacterial community. In summary, around 13 bands for the total bacterial community were systematically detected over long-term operation of the biofiltration system. (Samples 1a to 3a correspond to days 5, 10 and 20th of operation. Sample 4a was obtained at day 45th of operation, when the inlet load was increased to 3000 ppmv. Samples from 5a to 8a were obtained in days 90, 110 130 and 150 of operation, respectively.) In view of the total bacterial community, the bands remained constant until variations in intensity appeared. In lane 4a, lower intensity bands revealed a weakening pattern, which suggest a decrease in certain types of bacteria, when the H2S concentration increased from 1500 ppm to 3000 ppm. Both the decrease in removal efficiency and the decrease in the microbial population could be explained by the toxicity of the extremely high H2S concentration. This factor was assumed to be responsible for the disappearance of some of the microbial species. Increased intensity

position of sampling used for DGGE analysis.

Fig. 2. H2S elimination capacities as a function of H2S inlet loadings in a biofilter operated at A) 85 sec EBRT and B) 31 sec EBRT. The points and solid lines represent the experimental data, and the dashed line (--) is the 100% removal line measured at both EBRTs.

For long operation times, the biofilter nearly eliminated all the H2S from the biogas stream. The H2S concentrations of the AD gas stream were previously diluted to maintain an inlet concentration of 1500 ppmv and to allow complete elimination (99% removal efficiency), but the high removal efficiency was maintained over 90 days and complete biodegradation of VFAs was also observed. Fifty days after the start-up period, a technical failure in the AD system blocked the feeding of the biofilter, no data was obtained during that time. After operation conditions were restored, an inlet H2S concentration was maintained at approximately 1500 ppmv from day 103 to 194 at an EBRT of 31 sec. Under these conditions, a removal efficiency of 95% was maintained for 90 days. Higher concentrations, around 3000 ppmv, caused a drop in the biofilter efficiency to 50% (Ramirez-Saenz et al., 2009). The biofilter was fed with the ADS gas stream every two weeks, which corresponded to the HRT of the AD system.

According to the stoichiometry of aerobic biological H2S oxidation (Eq. 1 and 2) and the sulfate determinations obtained between days 103 and 194 of the operational period of the biofilter, 51 to 60% of the H2S was completely oxidized to sulfate. These data are correlated with those reported by Fortuny et al., 2008 with respect to the H2S conversion to sulfate. The elimination of these compounds allowed the potential use of the biogas while maintaining the methane (CH4) content throughout the process.

#### **3.3 Microbial community characterization**

Samples from three different positions of the biofiltration system were taken to evaluate the spatial distribution of the microbial population. Figure 3 shows a picture of the biofiltration system and the positions where the samples were collected. The samples at the top of the reactor "a" correspond to the inlet of the biogas stream mixed with different air fluxes, samples "b" and "c" correspond to the middle part of the reactor and sample "d" was located at the bottom of the biofilter (outlet of the biogas stream).

**EC(g/m3h)**

Fig. 2. H2S elimination capacities as a function of H2S inlet loadings in a biofilter operated at A) 85 sec EBRT and B) 31 sec EBRT. The points and solid lines represent the experimental

0 50 100 150 200 250 300 350 400

100% removal

**h)**

**Inlet load (g/m3**

For long operation times, the biofilter nearly eliminated all the H2S from the biogas stream. The H2S concentrations of the AD gas stream were previously diluted to maintain an inlet concentration of 1500 ppmv and to allow complete elimination (99% removal efficiency), but the high removal efficiency was maintained over 90 days and complete biodegradation of VFAs was also observed. Fifty days after the start-up period, a technical failure in the AD system blocked the feeding of the biofilter, no data was obtained during that time. After operation conditions were restored, an inlet H2S concentration was maintained at approximately 1500 ppmv from day 103 to 194 at an EBRT of 31 sec. Under these conditions, a removal efficiency of 95% was maintained for 90 days. Higher concentrations, around 3000 ppmv, caused a drop in the biofilter efficiency to 50% (Ramirez-Saenz et al., 2009). The biofilter was fed with the ADS gas stream every two weeks, which corresponded to the HRT

According to the stoichiometry of aerobic biological H2S oxidation (Eq. 1 and 2) and the sulfate determinations obtained between days 103 and 194 of the operational period of the biofilter, 51 to 60% of the H2S was completely oxidized to sulfate. These data are correlated with those reported by Fortuny et al., 2008 with respect to the H2S conversion to sulfate. The elimination of these compounds allowed the potential use of the biogas while

Samples from three different positions of the biofiltration system were taken to evaluate the spatial distribution of the microbial population. Figure 3 shows a picture of the biofiltration system and the positions where the samples were collected. The samples at the top of the reactor "a" correspond to the inlet of the biogas stream mixed with different air fluxes, samples "b" and "c" correspond to the middle part of the reactor and sample "d" was

maintaining the methane (CH4) content throughout the process.

located at the bottom of the biofilter (outlet of the biogas stream).

**3.3 Microbial community characterization** 

data, and the dashed line (--) is the 100% removal line measured at both EBRTs.

0 50 100 150 200 250

**h)**

**Inlet load (g/m3**

100% removal

of the AD system.

0

50

100

150

**EC (g/m3h)**

200

250

Fig. 3. Biofiltration system coupled to an anaerobic digestion system. Arrows indicate the position of sampling used for DGGE analysis.

Additionally, the changes in the bacterial community were also determined by taking samples during long-term operation of the biofiltration system (Samples from 1a to 9a). The analysis was performed by a DDGE system using 16S rRNA as a bacteria-specific target for PCR amplification. Figure 4 shows an example of denaturing gradient gel DDGE (15% to 60%) from samples of different times of cultivation compared with the initial bacterial community. In summary, around 13 bands for the total bacterial community were systematically detected over long-term operation of the biofiltration system. (Samples 1a to 3a correspond to days 5, 10 and 20th of operation. Sample 4a was obtained at day 45th of operation, when the inlet load was increased to 3000 ppmv. Samples from 5a to 8a were obtained in days 90, 110 130 and 150 of operation, respectively.) In view of the total bacterial community, the bands remained constant until variations in intensity appeared. In lane 4a, lower intensity bands revealed a weakening pattern, which suggest a decrease in certain types of bacteria, when the H2S concentration increased from 1500 ppm to 3000 ppm. Both the decrease in removal efficiency and the decrease in the microbial population could be explained by the toxicity of the extremely high H2S concentration. This factor was assumed to be responsible for the disappearance of some of the microbial species. Increased intensity

Biogas Production and

Cleanup by Biofiltration for a Potential Use as an Alternative Energy Source 127

Once the number of bands that were similar or different between the two samples was determined, the similarity of the different samples was determined by calculating the Jaccard and Sorensen-Dice indexes. Two different aspects were analyzed: the similarity of the samples during the time of cultivation (lanes 1a to 9a) and the similarity at a different

Band/Lane 1a 2a 3a 4a 5a 6a 7a 8a 9a 2b 2c 2d A 1 1 1 1 1 1 1 1 1 1 1 1 B 1 1 1 1 1 1 1 1 1 1 1 1 C 0 0 1 0 1 1 1 0 0 0 0 0 D 0 1 1 1 1 1 1 1 1 1 1 0 E 0 1 1 1 0 1 1 1 1 1 1 0 F 1 1 1 1 1 1 1 1 1 1 1 1 G 1 1 1 1 1 1 1 1 1 1 1 1 H 1 1 1 1 1 1 1 1 1 1 1 0 I 1 1 1 1 1 1 1 1 1 0 1 0 J 1 1 1 0 1 1 1 1 1 1 1 0 K 1 1 1 1 0 0 1 1 1 1 1 0 L 1 1 0 1 0 0 0 1 0 0 0 0 M 1 0 1 0 0 0 0 0 0 0 0 0 N 1 0 1 0 1 1 0 1 0 0 0 1 O 1 0 1 0 1 1 1 0 0 0 0 1 P 0 1 1 0 1 1 1 1 0 0 0 1 Q 0 0 0 0 0 1 1 0 0 0 1 1 R 1 1 1 0 1 1 1 0 0 1 1 1 S 0 0 0 0 0 0 0 0 0 0 0 1 Total 13 13 16 10 13 15 15 13 10 10 12 10 Table 2. Matrix constructed using the DDGE gel containing different band patterns. Lanes 1a-8a correspond to samples at 8 different times of cultivation. Lanes 9a to 9d were samples

Regarding time of cultivation, the bands A, B and E to J constantly appeared in the microbial community, suggesting little change in the microbial populations during the operation of the biofilter. The most similar microbial communities were found in lanes 6a and 7a, with a Jaccard index of 0.875 and a Sorensen-Dice index of 0.933, which corresponded to the steady state of the biofiltration system at an average H2S inlet concentration of 1500 ppmv and a removal efficiency of 95%. In contrast, the least similarity was found between lanes 4a and 5a, 6a and 7a (Jaccar´s indexes of 0.438, 0.47 and 0.56, respectively). In lane 4a, the microbial community sample was exposed to an increased H2S concentration of 3000 ppmv. These data differed from those found by Maestre et al., 2010. These authors reported a wide phylogenetic diversity and showed that the initial populations became more specific, being

The similarity between the microbial communities along the biofilter was also calculated. For this purpose, the bands in lane 9a were compared with the bands in lanes 9b, 9c and 9d. Significant differences in the microbial population were observed at different lengths along

position in the reactor (lane 9a compared to 9b, 9c and 9d).

at different lengths of the biofiltration system.

the SOB the dominant community.

in some bands in the gel (boxes A and B) demonstrated intensifications of specific band patterns. These data suggested the eventual dominance of H2S-oxidizing bacteria (SOB) and bacteria able to consume VFA.

Fig. 4. Polyacrylamide denaturing gradient gel (15–60%) with DGGE profiles of 16 S rRNA gene fragments of the samples taken from different operation times (Lanes from 1a to 8a) and locations of the biofilter (Lanes from 9a, inlet to 9d, outlet), (6-h run, 200 V, 60 °C).

To compare bacterial community between different samples and to determine possible changes in composition, the presence or absence of a band in a DDGE gel was analyzed using a binary system. A 0 value was assigned when the band was absent (i.e., different band is considered a different microorganism) and 1 when the band in two or more samples was present (i.e., same microorganism) at similar positions in the gel. Jaccard's index and the Sorensen-Dice index could then be calculated. Table 2 shows the matrix constructed using the DDGE gel containing different band patterns obtained at different times of operation (lanes 1a to 9a) and at different lengths along the biofilter (9a, 9b, 9c and 9d). Nineteen different bands (arbitrarily named A to S) were found in the samples analyzed by gradient DDGE.

in some bands in the gel (boxes A and B) demonstrated intensifications of specific band patterns. These data suggested the eventual dominance of H2S-oxidizing bacteria (SOB) and

Fig. 4. Polyacrylamide denaturing gradient gel (15–60%) with DGGE profiles of 16 S rRNA gene fragments of the samples taken from different operation times (Lanes from 1a to 8a) and locations of the biofilter (Lanes from 9a, inlet to 9d, outlet), (6-h run, 200 V, 60 °C).

To compare bacterial community between different samples and to determine possible changes in composition, the presence or absence of a band in a DDGE gel was analyzed using a binary system. A 0 value was assigned when the band was absent (i.e., different band is considered a different microorganism) and 1 when the band in two or more samples was present (i.e., same microorganism) at similar positions in the gel. Jaccard's index and the Sorensen-Dice index could then be calculated. Table 2 shows the matrix constructed using the DDGE gel containing different band patterns obtained at different times of operation (lanes 1a to 9a) and at different lengths along the biofilter (9a, 9b, 9c and 9d). Nineteen different bands (arbitrarily named A to S) were found in the samples analyzed by

bacteria able to consume VFA.

gradient DDGE.

Once the number of bands that were similar or different between the two samples was determined, the similarity of the different samples was determined by calculating the Jaccard and Sorensen-Dice indexes. Two different aspects were analyzed: the similarity of the samples during the time of cultivation (lanes 1a to 9a) and the similarity at a different position in the reactor (lane 9a compared to 9b, 9c and 9d).


Table 2. Matrix constructed using the DDGE gel containing different band patterns. Lanes 1a-8a correspond to samples at 8 different times of cultivation. Lanes 9a to 9d were samples at different lengths of the biofiltration system.

Regarding time of cultivation, the bands A, B and E to J constantly appeared in the microbial community, suggesting little change in the microbial populations during the operation of the biofilter. The most similar microbial communities were found in lanes 6a and 7a, with a Jaccard index of 0.875 and a Sorensen-Dice index of 0.933, which corresponded to the steady state of the biofiltration system at an average H2S inlet concentration of 1500 ppmv and a removal efficiency of 95%. In contrast, the least similarity was found between lanes 4a and 5a, 6a and 7a (Jaccar´s indexes of 0.438, 0.47 and 0.56, respectively). In lane 4a, the microbial community sample was exposed to an increased H2S concentration of 3000 ppmv. These data differed from those found by Maestre et al., 2010. These authors reported a wide phylogenetic diversity and showed that the initial populations became more specific, being the SOB the dominant community.

The similarity between the microbial communities along the biofilter was also calculated. For this purpose, the bands in lane 9a were compared with the bands in lanes 9b, 9c and 9d. Significant differences in the microbial population were observed at different lengths along

Biogas Production and

Cleanup by Biofiltration for a Potential Use as an Alternative Energy Source 129

concentration of 5 g/L. Under these conditions, conversion of thiosulfate to sulfate was stoichiometric, and the pH of the medium decreased from 8.0 to 6.6. The distance matrix phylogenetic tree based on the level of difference between *Bosea thiooxidans* and 19 reference strains of the alpha subclass of the Proteobacteria indicated that strain BI-42 belonged to a new lineage located between the methylotrophs, the genus *Beijerinckia*, and the *Rhodopseudomonas palustris* group. No close relationship was found between the strain and other sulfur-oxidizing bacteria, such as *Thiobacillus acidophilus* and *Acidiphilium* species (Das et al., 1996). Microorganisms utilize sulfur compounds for the biosynthesis of cellular material or transform these compounds as part of a respiratory energy-generating process. Most of the known sulfur-oxidizing bacteria belong to the genera *Thiobacillus*, *Thiothrix*, *Beggiatoa*, *Thiomicrospira*, *Achromatium*, *Desulfovibrio*, *Desulfomonas*, *Desulfococcus*, and *Desulfuromonas* . Furthermore, members of the genus *Thiobacillus* have been studied extensively to increase understanding of the coupling of oxidation of reduced inorganic

sulfur compounds to energy biosynthesis and assimilation of carbon dioxide.

Fig. 5. Neighbor-joining tree of partial 16S rRNA sequences (approximately 750 bp)

indicates 1% sequence variation.

recovered by denaturing gradient gel electrophoresis (DGGE) bands in the biofilter. The bar

The presence of *Arcobacter butzleri*, belonging to the Phylum Proteobacteria (e-Proteobacteria), could be associated with VFA degradation (these compounds were introduced into the biofilter through the biogas stream). This microorganism is able to grow under both aerobic and anaerobic conditions over a wide temperature range (15–42 °C). However, optimal growth occurs under microaerobic conditions (3–10% O2). *Arcobacter* 

the biofilter. The highest divergence was found between lanes 9d and 9b, with a Jaccard index of 0.333 and a Sorensen-Dice index of 0.5. These data could be partially explained by the H2S concentration gradient: a higher concentration at the inlet of the biofilter and a lower concentration at the outlet (sample 9d). The accumulation of metabolic products could also explain the divergence. The highest similarity was found in samples of lanes 9b and 9c, which corresponded to the middle of the reactor, where apparently the environmental conditions were more homogeneous (Jaccard index of 0.833 and Sorensen-Dice index of 0.909). These data are in agreement with the results obtained by Maestre et al., 2010 and Omri et al., 2011 about the divergence in microbial populations along the reactor.

Sequence analysis of DNA extracted from single bands representing specific species were then used as an approach for further community characterization. Sequence analyses of bands (Table 3) revealed the predominant bacteria in the biofiltration system. The structure of the bacterial community sequenced was associated with microbial activity in the system as a function of the pollutant eliminated in the biofiltration system.


Table 3. Sequence analysis and species identification of the major (7) DGGE bands for the biofilter samples.

Band sequencing results showed that the dominant members of SOB consisted of *Bosea thiooxidans* and *Thiobacillus* sp (Table 3)*.* Das et al., 1996 reported that *Bosea thiooxidans* was a new gram-negative bacterium isolated from agricultural soil and capable of oxidizing reduced inorganic sulfur compounds. Data showed that this microorganism was strictly aerobic. Experiments conducted to evaluate thiosulfate oxidation showed that the growth yield varied with the concentration of this compound; the greatest growth was observed at a

the biofilter. The highest divergence was found between lanes 9d and 9b, with a Jaccard index of 0.333 and a Sorensen-Dice index of 0.5. These data could be partially explained by the H2S concentration gradient: a higher concentration at the inlet of the biofilter and a lower concentration at the outlet (sample 9d). The accumulation of metabolic products could also explain the divergence. The highest similarity was found in samples of lanes 9b and 9c, which corresponded to the middle of the reactor, where apparently the environmental conditions were more homogeneous (Jaccard index of 0.833 and Sorensen-Dice index of 0.909). These data are in agreement with the results obtained by Maestre et al., 2010 and

Sequence analysis of DNA extracted from single bands representing specific species were then used as an approach for further community characterization. Sequence analyses of bands (Table 3) revealed the predominant bacteria in the biofiltration system. The structure of the bacterial community sequenced was associated with microbial activity in the system

Table 3. Sequence analysis and species identification of the major (7) DGGE bands for the

Band sequencing results showed that the dominant members of SOB consisted of *Bosea thiooxidans* and *Thiobacillus* sp (Table 3)*.* Das et al., 1996 reported that *Bosea thiooxidans* was a new gram-negative bacterium isolated from agricultural soil and capable of oxidizing reduced inorganic sulfur compounds. Data showed that this microorganism was strictly aerobic. Experiments conducted to evaluate thiosulfate oxidation showed that the growth yield varied with the concentration of this compound; the greatest growth was observed at a

biofilter samples.

**Band No.** 

**Closest relative** 

*mediolanus* <sup>100</sup>

*butzleri* 99%

*thiooxidans* 94%

*fibrisolvens* 99%

*bacteria* 98%

100%

3 *Bacillus cereus* 98%

<sup>1</sup>*Agromyces* 

<sup>2</sup>*Arcobacter* 

<sup>4</sup>*Bosea* 

<sup>5</sup>*Butirivibrio* 

<sup>6</sup>*Thiobacillus sp.* 

<sup>7</sup>*Uncultured* 

**Identity (%)** 

Omri et al., 2011 about the divergence in microbial populations along the reactor.

as a function of the pollutant eliminated in the biofiltration system.

concentration of 5 g/L. Under these conditions, conversion of thiosulfate to sulfate was stoichiometric, and the pH of the medium decreased from 8.0 to 6.6. The distance matrix phylogenetic tree based on the level of difference between *Bosea thiooxidans* and 19 reference strains of the alpha subclass of the Proteobacteria indicated that strain BI-42 belonged to a new lineage located between the methylotrophs, the genus *Beijerinckia*, and the *Rhodopseudomonas palustris* group. No close relationship was found between the strain and other sulfur-oxidizing bacteria, such as *Thiobacillus acidophilus* and *Acidiphilium* species (Das et al., 1996). Microorganisms utilize sulfur compounds for the biosynthesis of cellular material or transform these compounds as part of a respiratory energy-generating process. Most of the known sulfur-oxidizing bacteria belong to the genera *Thiobacillus*, *Thiothrix*, *Beggiatoa*, *Thiomicrospira*, *Achromatium*, *Desulfovibrio*, *Desulfomonas*, *Desulfococcus*, and *Desulfuromonas* . Furthermore, members of the genus *Thiobacillus* have been studied extensively to increase understanding of the coupling of oxidation of reduced inorganic sulfur compounds to energy biosynthesis and assimilation of carbon dioxide.

Fig. 5. Neighbor-joining tree of partial 16S rRNA sequences (approximately 750 bp) recovered by denaturing gradient gel electrophoresis (DGGE) bands in the biofilter. The bar indicates 1% sequence variation.

The presence of *Arcobacter butzleri*, belonging to the Phylum Proteobacteria (e-Proteobacteria), could be associated with VFA degradation (these compounds were introduced into the biofilter through the biogas stream). This microorganism is able to grow under both aerobic and anaerobic conditions over a wide temperature range (15–42 °C). However, optimal growth occurs under microaerobic conditions (3–10% O2). *Arcobacter* 

Biogas Production and

1879.

*Bioproducts and Biorefining*, 3, 42-71.

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Abatzoglou, N. & Boivin, S. (2009). A review of biogas purification processes. *Biofuels,* 

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Alvarez, R. (2004). Produccion anaerobica de biogas, aprovechamiento de los residuos del

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Angelidaki, I., Ellegaard, L., Ahring B.K. (2003). Applications of the anaerobic digestion

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Bouallagui, H., Lahdheb, H., Ben Romdan, E., Rachdi, B., Hamdi, M. (2009). Improvement of

Chung, Y.C., Huang, C., Tseng, C.P. (1996). Operation and optimization of *Thiobacillus*

Chung,Y.C. (2007(. Evaluation of gas removal and bacterial community diversity in a

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substrates addition. *Journal of Environmental Management*, 90, 1844-1849. Bouallagui, H., Touhami, Y., Ben Cheikh, R., Hamdia, M. (2005). Bioreactor performance in

Daffonchio, D. (2006). Microbial succession in a compost-packed biofilter treating

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*butzleri* was also recently found as a member of the microbial population in a microbial fuel cell (MFC) used to produce electricity from synthetic domestic wastewater that contained a mixture of VFAs as electron donors (Freguia et al., 2010). Similar results were obtained by Nien et al., 2011, where an *Arcobacter butzleri* strain, ED-1, was also determined to be part of the microbial community of a MFC fed with acetate. Although aerobic species were predominant because the metabolic activity determined (sulfate as the main product), the DGGE showed that profile some facultative anaerobes were as part of the microbial population, which could be related to the trophic properties of the community, and the different substrates in the biogas stream (H2S and VFAs).

Some of the species found in the present study agreed with those previously reported in the literature for biofiltration systems used in the removal of reduced sulfur compounds. For example, Ding et al., 2006 studied a packed compost biofilter for the treatment of a mixture of H2S and methanol using 16S rRNA sequencing analysis. The authors established that the microbial community was composed of strains of *Thiobacillus*, *Sulfobacillus*, and *Alicyclobacillus hesperidensis*. In a biofilter packed with compost, activated carbon and sludge used for the removal of H2S, Chung, 2007 determined a microbial population composed of *Pseudomonas citronellolis*, *P. fluorescens*, *P. putida*, *S. capitis*, *Bacillus subtilis* and *Paracoccus denitrificans*. In a recently published work (Omri et al., 2011), it was reported that most bacteria in the operation samples were of the genera *Pseudomonas* sp., *Moraxellacea*, *Acinetobacter* and *Exiguobacterium*, which belong to the phyla Pseudomonadaceae, gamma-Proteobacteria and Firmicutes.

A neighbor-joining tree (Fig 5.) of partial 16S rRNA sequences (approximately 750 bp) was constructed in MEGA4 (Tamura et al., 2007) by considering sequences obtained and comparing them with others in the data bank.
